Cells integrate information from their constituents and encode the normal and abnormal traits of life. The temporal-spatial interplay of ions, metabolites, macromolecules, and organelles are responsible for carrying out the complex reactions that perform life functions. It has recently become evident that normal cell functions and those associated with a cancerous state involve changes in the microscopic distribution of cellular constituents, in addition to their activity. Therefore, an understanding of the role of selected targets in cell functions requires tools to automatically extract temporal and spatial information about the target activities. High content screens (HCS) have been developed to address the need for more detailed information about the temporal-spatial dynamics of normal and transformed cell constituents. HCS automate the extraction of information derived from specific fluorescence-based reagents incorporated into cells. The concept is to treat each cell as a "well" that has spatial and temporal information on the activities of the labeled constituents. HCS will permit efficient lead optimization before the time consuming and expensive animal testing stage. The overall goal of this Project is to design and implement HCS that augment the in vitro activity assays presented in Projects 2 and 3 and to provide the cell- based structure-activity data for lead optimization. To test the hypothesis that complex physiological responses to drug treatments can be dissected using HCS of specific cellular and molecular processes, the following is proposed: 1) To develop HCS of apoptosis that simultaneously measures drug-induced changes in cell and nuclear morphology, DNA content, actin-cytoskeletal assembly, and plasma membrane lipid dynamics within single tumor cells; 2) To develop HCS of drug-induced temporal-spatial dynamics of the microtubule cytoskeleton within single living tumor cells using a transiently expressed green fluorescent protein (GFP)-microtubule associated protein chimera as a reporter of intracellular microtubule assembly; and 3) To develop HCS to measure drug-induced temporal-spatial dynamics of distribution and activity of dual specificity phosphatases by engineering cdc25 molecules into fluorescent protein biosensors.